Figure 4

Minodronate and alendronate inhibited osteoclast formation via suppression of GGPP biosynthesis in RAW264.7 cells. RAW264.7 cells were pretreated with 20 μM FOH or 20 μM GGOH for 4 h and then treated with 10 μM minodronate or 30 μM alendronate, and 50 ng/mL RANKL for 7 days. Cultures were fed every 2 days by replacing with 500 μL of fresh medium, with or without minodronate, alendronate, RANKL, FOH, and GGOH. (A, B) Inhibitory effect of mevalonate pathway intermediates FOH or GGOH on the inhibition of osteoclast formation by minodronate (A) or alendronate (B). Cultures were fixed and stained for TRAP-positive multinucleated cells, and the number of cells per well was counted. These results are representative of 5 independent experiments. *P < 0.01 compared to 50 ng/mL RANKL administration. (C) CTR and cathepsin K mRNA expression in RAW264.7 cells that were treated with minodronate or alendronate along with FOH or GGOH. Total RNA was extracted, and the CTR and cathepsin K mRNA levels were determined by real-time PCR. The results are expressed as the ratio of treated to control samples after normalization to GAPDH mRNA levels. The results are representative of 4 independent experiments. *P < 0.01 compared to 50 ng/mL RANKL administration. (D, E) Cultures were fixed and stained for TRAP-positive cells, and the number of cells per well was counted. These results are representative of 4 independent experiments. *P < 0.01 compared to 50 ng/mL RANKL administration.